Scattered light integrating collector

ABSTRACT

A system for measuring a liquid sample comprising biological material, the system comprising an integrating light collector for collecting light and for at least partially containing the sample; a light source for introducing light in the integrating light collector; a signal generator or modulator configured to cause a known modulation of the light output by the light source; a phase-sensitive detector for detecting scattered light in the integrating light collector; at least one of an exit port to allow un-scattered light to exit the integrating light collector, a beam dump, or a baffle arranged to absorb unscattered light; and a processor configured to analyse the detected modulated light to determine changes in the detected modulated light as a function of time thereby to determine at least one of: drug susceptibility of the biological material; a change in a number of cells in the sample; a change in cell state; a change in the biological material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/549,507, filed Aug. 8, 2017, which is a national stage applicationunder 35 U.S.C. 371 and claims the benefit of PCT Application No.PCT/GB2016/050319 having an international filing date of 10 Feb. 2016,which designated the United States, which PCT application claimed thebenefit of Great Britain Application No. 1502194.2 filed 10 Feb. 2015,the entire disclosures of each of which are incorporated herein byreference.

The present invention relates to a device for measuring at least oneproperty of a sample, for example a biological sample such as bacteria,using light.

BACKGROUND OF THE INVENTION

Classic spectrophotometers can be used to determine optical propertiesof bacteria using absorption or scattering. Absorptionspectrophotometers can be used to measure the relative absorbance of asample. Absorbance is measured by comparing the intensity of lightentering a sample with the intensity of light exiting the sample. A dropin light intensity indicates a quantity of light has been absorbed. Thiscan be displayed as an arbitrary figure, typically an optical density.This can lead to an accurate count of the number of cells present in asample.

Scattering spectrophotometers usually comprise an intense light source,such as a laser or a very bright incandescent source, and amonochromator. Light is incident on a sample and is scattered atdifferent angles. Detectors placed at discrete intervals around achamber collect the scattered light. Collected light in the sidescattering region can be used to obtain information about granularityand light collected in the forward scattering region can be used toobtain information about the size of the particles. Overall intensity ofthe scattered light gives a turbidity reading and an indication of thenumber of particles present. In scattering spectrophotometers formeasuring bacteria, the typical wavelength of the light source is 600nm. This wavelength is the most scattered and least absorbed by a numberof organic materials, such as DNA, proteins, cytochromes.

Flow cytometers can also determine properties of a sample of interest.When a sheath-flow of index matched liquid flows through a narrow tube,the liquid acts to reduce the lumen of the tube forcing cells in theliquid to pass through the tube individually. This facilitates cellcounting. Laser light incident on the narrow tube is scattered asindividual cells pass through. Side and forward scattering data can berecorded to give information about the size and granularity of the cellsunder study. Thousands of cells can pass through the beam and bemeasured in this way in a few seconds and in very little liquid. Whilstcytometers are useful in some applications, they are sophisticatedmachines that require extensive training of an operator. Safe operationalso requires a regular input of reagents and this contributes toon-going running costs. The interpretation of data produced can alsoprove challenging.

Another method for measuring concentration of suspended particles in aliquid or gas is nephelometry. Nephelometers can be configured to useintegrating spheres. In such a configuration, light is incident on asample and may be scattered by particles in the sample before enteringthe integrating sphere. The scattered light is then reflected anddiffused inside the integrating sphere before being detected at an exitport of the sphere. Unscattered light passes straight through the sphereand is not collected.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided adevice comprising an integrating sphere light collector for collectinglight and adapted to contain a sample within its internal volume in use.A sample holder may be provided to hold a sample inside the integratingsphere light collector. Typically, the sample comprises a fluid.Preferably a detector is provided on an internal surface of theintegrating sphere light collector. A baffle may be positioned over thedetector to prevent its direct illumination and ensure that onlyscattered and reflected light is incident on it.

By providing a sample holder that locates a sample inside an integratingsphere, highly sensitive measurements can be taken. This is because thehollow spherical cavity of the integrating sphere acts as a lightdiffusion and collection chamber. Light inside the cavity is reflectedmultiple times off the internal surface to produce a uniformdistribution of light throughout the interior of the cavity. Because thesample is located inside the hollow spherical cavity, beams of light maypass through it multiple times.

The sample holder may comprise an internal volume of the integratingsphere. In this case, the internal volume is flooded with the sample andfulfils a dual purpose, i.e. to contain the sample and to act to diffuseand collect light. The sample may substantially fill the interior of theintegrating sphere.

The integrating sphere light collector comprises an entry port forallowing light to enter the integrating sphere light collector and anexit port to allow unscattered light of the incident beam to exit theintegrating sphere light collector.

The sample holder may be adapted to hold a sample substantiallycentrally within the integrating sphere light collector. The sample maybe within a sample holder that extends along a diameter of theintegrating sphere.

The sample holder may be adapted to hold a cuvette, for example astandard 10 mm length cuvette, in which a sample can be loaded. Morespecifically, the holder may be adapted to hold a cuvette of dimensions45 mm×10 mm×10 mm (square footprint).

The sample holder may be a flow through holder for allowing a sample toflow through the integrating sphere light collector.

According to another aspect of the invention, there is provided a systemfor analysing a sample comprising: an integrating sphere light collectorfor collecting light and containing the sample; a light source forintroducing light in the integrating sphere light collector; a signalgenerator for generating a control signal to cause the light source tooutput modulated light; a detector for detecting scattered light in theintegrating sphere light collector and generating a signal indicative ofthe scattered light, and a lock-in amplifier operable to use a signalfrom the signal generator indicative of the light modulation and thesignal generated by the detector to provide an output for analysis.

The light source may comprise a laser or a LED. The light source may belocated in the integrating sphere light collector. The light source maybe located external to the integrating sphere light collector. The lightsource may have a wavelength in the range of 590 nm to 650 nm, forexample 635 nm. The light source may have a wavelength in the range of620 nm to 750 nm, for example 635 nm.

According to yet another aspect of the present invention there isprovided a device comprising an integrating sphere light collector forcollecting light and containing a sample, and at least one light sourceand at least one detector on an internal surface of the integratingsphere light collector. The integrating sphere light collector mayinclude a sample holder for containing the sample within its internalvolume. The sample holder may be adapted to hold a sample substantiallycentrally within the integrating sphere light collector. The sampleholder may be a flow through sample holder for allowing a sample to flowthrough the integrating sphere light collector. The sample holder may beadapted to hold a sample cuvette. The integrating sphere light collectormay have an exit port to allow unscattered light to exit the integratingsphere light collector. Unscattered beam suppression means may beprovided.

According to still another aspect of the present invention, there isprovided a device comprising an integrating sphere light collector forcollecting light and for containing a sample, and a pipette tip, whereinthe integrating sphere light collector is at one end of the pipette tipand the pipette tip is arranged to draw a sample fluid into theintegrating sphere light collector.

According to still another aspect of the present invention, there isprovided a method for monitoring drug susceptibility of a biologicalsample, the method comprising: introducing the biological sample into anintegrating sphere light collector; introducing a drug into thebiological sample; introducing light into the integrating sphere lightcollector, so that the light passes through and is scattered by thesample; detecting scattered light in the integrating sphere lightcollector; repeating the steps of emitting and detecting as a functionof time and analysing the detected light to determine drugsusceptibility. The sample may comprise a species or strain ofbacteria/fungi. Analysing the captured light may involve establishingthe level of drug that kills or inhibits growth of a given organism. Themethod may involve monitoring an undosed biological sample at the sametime as the drugged sample.

According to still another aspect of the present invention, there isprovided a method for counting cells, the method comprising: introducinga sample into an integrating sphere light collector; emitting light inthe integrating sphere light collector, so that the light passes throughand is scattered by the sample; detecting scattered light in theintegrating sphere light collector; and analysing the detected light todetermine the number of cells.

According to still another aspect of the present invention, there isprovided a method for determining a cell state of a bacterial culture,the method comprising: introducing a bacterial culture sample into anintegrating sphere light collector; emitting light in the integratingsphere light collector, so that the light passes through and isscattered by the sample; detecting scattered light in the integratingsphere light collector; and analysing the detected light to determinethe number of cells, wherein changes in the detected light as a functionof time are indicative of a change in cell state.

According to still another aspect of the present invention, there isprovided a method for monitoring a biological material, the methodcomprising: introducing a biological sample into an integrating spherelight collector; emitting light in the integrating sphere lightcollector, so that the light passes through and is scattered by thesample; detecting scattered light in the integrating sphere lightcollector; and analysing the detected light, wherein changes in thecaptured light as a function of time are indicative of a change in thebiological material. The change in the biological material may be achange in cell state.

The biological material may include a pathogen and the change in thebiological material may be a change in a level or concentration of thepathogen, thereby indicating growth of that pathogen.

The biological material may include a microbe and the change in thebiological material may be a change in a level or concentration of themicrobe, thereby indicating growth of that microbe.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the invention will now be described by way of exampleonly, and with reference to the accompanying drawings, of which:

FIG. 1 is schematic representation of an integrating sphere lightcollector for measuring optical properties of a sample, and inparticular a biological sample;

FIG. 2 is a block diagram of a detection and analysis system for usewith the integrating sphere light collector of FIG. 1;

FIGS. 3(a) and (b) show various parts of a first example integratingsphere light collector and sample holder;

FIGS. 4(a) to (c) show various parts of a second example integratingsphere light collector and sample holder;

FIGS. 5(a) to (c) show various parts of a third example integratingsphere light collector and sample holder;

FIG. 6 is a schematic representation of a fourth example integratingsphere light collector;

FIG. 7 is a plot of detector output as a function of time for two E.coli samples, one with a drug and one without;

FIG. 8 is a plot of detector output as a function of time for two S.marcescens samples, one with a drug and one without;

FIG. 9 is a plot of detector output as a function of time for two S.epidermidis samples, one with a drug and one without;

FIG. 10 is a top view of an integrating sphere light collector withinternal light sources;

FIG. 11 is a perspective view of a lower half of the integrating spherelight collector of FIG. 10;

FIG. 12 is a logarithmic plot of scattering intensity as a function oftime for different sample dilutions;

FIG. 13 is a logarithmic plot of scattering intensity as a function oftime for a single sample on which Mu is shown;

FIG. 14 is a plot of the inverse of Mu as a function of per division ofbacteria, and

FIG. 15 is a plot “number of divisions till positivity” vs number ofcells in a sample at a measurement start point.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scattered light integrating collector 10. The collector10 has an integrating sphere 12 and a sample holder 14 for holding asample within the integrating sphere 12. In the example shown in FIG. 1,the sample holder 14 is adapted to hold a sample cuvette 16 within thesphere 12.

The integrating sphere 12 has a hollow spherical cavity, an entry port18 and an exit port 20. The entry port 18 and exit port 20 define theend points of an optical path through the hollow spherical cavity. Theentry and exit ports 18 and 20 respectively are positioned on opposingsides of the spherical cavity. An internal surface of the hollowspherical cavity is diffusive and so capable of reflecting and diffusinglight. In some cases, a thin aluminium or silver coating is applied toan inner surface of the sphere and covered with a layer of titaniumoxide II paint. These layers reflect back any laser radiation that wasscattered by the sample and diffuse any light that was scattered by thesample and reaches the inner face of the sphere, respectively.

The sample holder 14 and sample cuvette 16 are positioned so that in usethe sample extends across substantially the entire diameter of theintegrating sphere 12. This helps maximise the volume of the sample thatcan interact with reflected and diffused light circulating within thesphere.

On the internal surface a photodetector 22 is provided, for example aphotodiode. This is used to measure the intensity of light in the cavityas a function of time. A baffle is positioned over the photodiode toprevent its direct illumination and ensure that only scattered andreflected light is incident on it, thereby increasing the quality of thesignal.

Light enters the hollow spherical cavity of the integrating sphere 12through the entry point 18. The hollow cavity acts as a light diffusionand collection chamber. Light inside the cavity is reflected multipletimes off the internal surface of the hollow cavity to produce a uniformdistribution of light throughout the interior of the cavity. Unscatteredlight exits the hollow cavity through the exit port 20 to a beam dump.Scattered light is measured by the photodetector 22. Because the sampleis located inside the hollow spherical cavity, beams of light may passthrough it multiple times. This results in highly sensitivemeasurements.

FIG. 2 shows a detection and analysis system for use with the collectorof FIG. 1. At the input port 18 of the collector is a light source 24,for example is a 635 nm wavelength laser (although any red light in thewavelength 620-750 nm could be used), for inputting light to theintegrating sphere 12. The laser 24 is connected to a signal generator26 that is adapted to control a modulation frequency and phase of thelaser output. The photodiode 22 is connected to a lock-in amplifier 28.An input of the amplifier 28 is connected to the signal generator 26. Anoutput of the amplifier 28 is connected to a digital oscilloscope 30.The lock-in amplifier 28 uses phase-sensitive detection to single out acomponent of the signal at a specific reference frequency and phase, inthis case the modulation frequency that is set by the signal generator.Noise signals, at frequencies other than the reference frequency, arerejected and do not affect the measurement. An output from the digitaloscilloscope 30 is fed to a computer display 32.

The signal generator 26 is arranged to modulate the output frequency ofthe laser source 24. As an example, the laser may be modulated at afrequency of 10 kHz with a phase of +169°, and a peak-to-peak amplitudeof 200 mV. The detected signal is filtered by the lock-in amplifier 28.The lock-in amplifier 28 filters the detected signal from the photodiode22. The lock-in amplifier 28 synchronizes the detected signal with themodulation applied to the light source 24 to provide a dampening systemthat eliminates unwanted noise, for example, background electrical orluminous noise. The filtered signal is sent to the digital oscilloscope30 to be recorded. The recorded signal can be displayed on the computerdisplay 32.

Raw data is collected by the digital oscilloscope. Typically around16,000 data points are collected for every 30 second experiment. Thedata is exported to a calculation suite in a processor which returns theaverages (mean, median, mode) and the standard deviation of the datapoints. If the standard deviation is above a threshold (indicatingaberrations from the norm in the data) the data is discarded. The meanof each experiment is selected. The experiments have between 3 and 89technical replicates, which are collected and tabulated. The standarderror from the mean of these averages is calculated and charted as errorbars along with the data. Once the data is graphed, a function, such asa standard Gompertz, is fitted to the data in order to estimate futureoutcomes of experiments such as inocula sizes.

In use, a sample is placed inside the sample cuvette 16 and positionedin the sample holder 14, which holds the sample in the interior of thehollow cavity. Incoming light from the source 24 enters the cavitythrough the entry port 18. The sample is positioned such that theincoming light beam is incident on the sample. Incoming light may bescattered by the sample. The scattered light is then reflected, multipletimes, by the internal surface of the cavity. The hollow cavity acts asan integrating sphere and integrates or adds up the reflected lightinside the sphere. The sum of the diffused light is sampled by thephotodiode 22. This is done as a function of time. Unscattered lighttravels straight through the cavity and is absorbed by a beam dump or abaffle.

Due to the geometry and scattering properties of the internal surface ofthe hollow cavity of the integrating sphere 12 reflected light isincident on the sample from all directions. With a sample present insidethe hollow cavity, the distribution of the light detected by thephotodetector 22 will change dependent on the optical properties of thesample.

Various different embodiments of the integrating sphere 12 with itsinternal sample arrangement will now be described.

FIG. 3(a) shows two parts that make up the spherical portion of acollector. FIG. 3(a)(i) shows an upper part of the collector with anupper hemisphere. The upper part of the collector has a sample port forreceiving a sample holder. FIG. 3(a)(ii) shows a lower part of thecollector which has a base and a lower hemisphere. In this part, a portis provided for holding a photodiode. This is positioned behind abaffle, so that only scattered or reflected light is transmitted to thephotodiode. FIG. 3(b) shows two parts of a sample holder for insertingin the sample port of FIG. 3(a)(i). The sample holder has a lid and abody for holding the sample, and in particular in this case, a samplecuvette. When assembled the sample body is designed to hold the sampleat the centre of the collector. The lid and body connect together toform a sample holder. The integrating sphere is formed by connecting theupper and lower hemispheres of FIG. 3(a). A sample is loaded into thesample holder, which is then loaded into the sample port of thecollector. The sample holder holds the sample in the centre of thecollector. The lid of the sample holder completes the sphere of thecollector.

FIGS. 4(a), 4(b) and 4(c) show parts of another example collector. FIG.4(a) shows a lower half of a collector. The lower half is a hemispheremodified to contain part of a sample port. FIG. 4(b) shows the upperhalf of the collector. FIG. 4(c) shows a sample holder. The sampleholder has a curved surface that completes the interior of the collectorwhen assembled. In use, the two halves of the collector are connectedtogether leaving an open sample port. The sample holder of FIG. 4(c) isthen inserted into the sample port, which completes the integratingsphere and locates the sample in a central position inside the sphere.

FIGS. 5(a), 5(b) and 5(c) show parts of yet another example collector.In this case, the collector is formed from blocks of material withhollowed out cavities. FIG. 5(a) shows a lower block. The lower blockhas a hollow hemispherical cavity formed in it, and a sample sitelocated at the base of the cavity. The lower block is adapted to house alaser. FIG. 5(b) shows the upper block. The upper block has a hollowhemispherical cavity formed in it. FIG. 5(c) shows a laser casing forconnecting to the lower block. In use, a sample is loaded on to thesample site. The upper block is then attached thus completing a hollowspherical cavity. The laser casing is removed from the lower block onlyfor maintenance of the laser.

In all of the examples described with reference to FIGS. 3 to 5, thesample holder is designed to minimise interference with the lightcirculating in the sphere. For example, the sample holder may be made ofa material substantially transparent at the wavelength of operation.

FIG. 6 show a pipette-like device that incorporates a sphericalcollector. The spherical collector is an integrating sphere 12 asdescribed above. The device has a pipette tip 34 and a collector 12. Thepipette tip 34 is disposable. As before, in the wall of the collector isa photodiode 22. The interior of the spherical collector acts as asample chamber. In use, a sample is drawn up through the pipette tip 34by a pipette mechanism. The sample is drawn into the sample chamber, sothat the interior of the spherical collector is flooded with the sample.Once the sample is present inside the spherical collector, a laser canbe activated. Light scattered by the sample, and reflected by thecollector is then detected by the photodiode 22 embedded in the wall.After a reading is made, the contaminated device can be disposed.

The device of the invention can be used to determine bacterialsusceptibilities to drugs. This is done over time with a setconcentration of drug. To do this, bacterial species are measured anddiluted or concentrated to a clinically significant level. A quantity ofdrug that the bacteria are susceptible to is added at a concentrationgreater than the accepted MIC (minimum inhibitory concentration). Thedosed culture is grown at accepted conditions in parallel with anotherculture that has been treated identically with the exclusion of thedrug. The diluent used for the drug (PBS or water) is added at the samevolume as the drug in the dosed culture. At predetermined time points,the cultures are removed from the incubator and measured in theintegrated collector in 1 ml cuvettes. The first time point at whichthere is a statistically significant difference between the dosed andthe free-growing cultures is declared the time to positivity (TTP).Tests have demonstrated that the system of the invention has a TTPfaster than any other drug susceptibility apparatus on the market.

Various drug susceptibility experiments have been conducted. For these,the collector of FIG. 3 was used. The oscilloscope used was a picoscope4226 with picoscope software to translate the raw data. The light sourceused was a modulated diode laser with a well-defined output wavelengthof 635 nm. The scan rates for the oscilloscope (which is the ratelimiting stage) are around 200 Hz. It is set to take a measurement everymillisecond (1 kHz). However, the processor used limited the averageamount of data to around 1600 data points per 30 second experiment. Thisworks out at 0.01875 measurements per second or around 200 Hz. Data fromthe oscilloscope is imported into data handling software (e.g. excel, R,SPSS, matlab) and the average of the ˜1600 data points from the 30second scan is calculated. The standard deviation and/or standard errorcan be calculated and used to show the stability of the signal duringthe scan.

FIG. 7 is a graph of detector output (in mV) as a function of time fortwo E. coli samples, one with a drug and one without, from threeseparate experiments in which triplicates of each sample were run (n=9).Error bars are plus and minus one standard error from the mean. The blueline indicates the sample in which 20 μg/ml of ciprofloxacin was addedprior to inoculation with bacteria. Bacteria were added at levels underthe detection limit of a Shimadzu UV-1601 UV-Vis spectrophotometer andlater quantified by CFU count as between 300-700 cells/ml across allexperiments. The red line indicates the sample that had no drug addedand was allowed to grow normally with the same number of bacteria addedat the same time point. Cultures were incubated at 37° C. shaking at 210RPM between sampling and sampling was limited to between 3-5 minutes toarrest any loss of heat from the samples so as to not greatly affecttheir growth times. Statistical tests (T test and Chi squared) indicatethe 30-minute time point to be first point at which there is asignificant difference between the two samples. Therefore, 30 minutes isthe detection time.

TABLE 2 Chi squared and T-test results for all time points in FIG. 7.minutes 0 15 30 45 60 Chi² 0.934147 0.180333 0.024681 6.5E−05 9.33E−10T-test 0.207025 0.034152 0.004411 0.000271 0.002039

FIG. 8 is plot of detector output (in mV) as a function of time for twoS. macresens samples, one with a drug and one without, from threeseparate experiments in which triplicates of each sample were run (n=9).Error bars are standard error from the mean. The blue line indicates thesample in which 20 μg/ml of ciprofloxacin was added prior to inoculationwith bacteria. Bacteria were added at levels under the detection limitof a Shimadzu UV-1601 UV-Vis spectrophotometer and later quantified byCFU count as between 300-700 cells/ml across all experiments. The redline indicates the sample that had no drug added and was allowed to grownormally with the same number of bacteria added at the same time point.Cultures were incubated at 37° C. shaking at 210 RPM between samplingand sampling was limited to between 3-5 minutes to arrest any loss ofheat from the samples so as to not greatly affect their growth times.Statistical tests (t-test and Chi squared) indicate the 30 minute timepoint to be first point at which there is a significant differencebetween the two samples. Therefore, 30 minutes is the detection time.

TABLE 3 Chi squared and T-test results for all time points in FIG. 8.minutes 0 15 30 45 60 Chi² 0.835984 0.239163 0.039273 0.005643 0.000106T-Test 0.298238 0.03116 0.007687 0.010741 0.006206

FIG. 9 is a plot of detector output as a function of time for two S.epidermidis samples, one with a drug and one without, from threeseparate experiments in which triplicates of each sample were run (n=9).Error bars are standard error from the mean. The blue line indicates thesample in which 20 μg/ml of ciprofloxacin was added prior to inoculationwith bacteria. Bacteria were added at levels under the detection limitof a Shimadzu UV-1601 UV-Vis spectrophotometer and later quantified byCFU count as between 300-700 cells/ml across all experiments. The redline indicates the sample that had no drug added and was allowed to grownormally with the same number of bacteria added at the same time point.Cultures were incubated at 37° C. shaking at 210 RPM between samplingand sampling was limited to between 3-5 minutes to arrest any loss ofheat from the samples so as to not greatly affect their growth times.Statistical tests (T test and Chi squared) indicate the 30-minute timepoint to be first point at which there is a significant differencebetween the two samples. Therefore, 30 minutes is the detection time.

TABLE 4 Chi squared and T-test results for all time points in FIG. 9.minutes 0 15 30 45 60 Chi² 0.893886 0.520936 0.017505 8.32E−06 3.71E−09T-test 0.445398 0.019607 0.001156 0.020599 0.012607

FIGS. 7 to 9 indicate that at 30 minutes there is a significantdifference between the sample dosed with ciprofloxacin and the oneallowed to grow normally. A detection time of 30 minutes is asignificant improvement on the detection times of known technology.

The experiments described above may be extended for a clinical lab toallow many samples to be tested simultaneously. Cultures with suspectedbacterial growth (blood samples from sepsis for example) need simply beloaded into blood incubator tubes (as is done now in hospitals) and havesuspected efficacious drugs added, one to each tube totalling, forexample, 20 tubes plus one control with no drug. These would all then begrown as is current standard procedure with samples removed and analysedby SLIC every 15-30 minutes until it is clear which drugs are effectivein retarding growth of the bacteria relative to the control.

In the experiments described above, the sample is held within a constantvolume sample container, i.e. a sample cuvette. It will be appreciatedthat the invention can be used in a constant flow system. For example, aflow cuvette may be placed in the spherical collector with feeding anddraining tubes attached. A bacterial culture may be passed through thecuvette by gravity pumping from a heated reservoir and measurementstaken constantly.

For a flow based system, the flow rate has to be controlled to ensurethat sufficient samples can be taken. The flow rate can be determinedusing:flow rate=¼×π×(pipleine diameter)²×velocity velocity=sampling rate×beamvolume

Using the oscilloscope and processor described above, with themeasurement frequency of 200 Hz, a flow pipeline diameter of 10 mm and abeam volume of 30 mm³, a flow system would require the flow rate to belimited to ˜470 ml/s (roughly half a litre per second). A fasterprocessor would speed up this system considerably.

Real-time growth curves can be collected using the device of theinvention. In this case, the device would be placed in an incubator witha static or flowing culture vessel within it. Data would be collectedover time, so that a sample could be measured for turbidity at any timepoint required. Indeed, the measurements could be taken multiple timesper minute or continuously.

FIG. 10 shows another embodiment of the invention. In this case, thedevice is adapted to perform fluorescence measurements using at leastone light source and at least one detector mounted inside theintegrating sphere, the at least one light source being operable to emitlight of at least one wavelength suitable for stimulating fluorescenceand the at least one detector being operable to detect the emittedfluorescence. For fluorescence measurements, the whole sample has to beilluminated and there is no need for a light exit port. To ensure thatonly fluorescence is detected a combination of a photodiode and anoptical shield/filter may be used as the detector. The shields may bemoulded from optical quality plastics at a specific wavelength bandpass.

FIG. 10 shows two light sources 36, in this case LEDs, and twoassociated light detectors 38, in this case photodiodes, are provided onan internal surface of an integrating sphere light collector. The lightsource(s) 36 emit at a wavelength matched to the absorptionwavelength(s) of the material(s) of interest. The photodiodes 38 havepeak sensitivity at the expected fluorescence emission range.

FIG. 11 shows a 3D rendering of the bottom half of a sphere that can beused to produce a physical 3D device using for example 3D printingtechniques. The wavelength of light emitted by the LEDs is selected tostimulate fluorescence of a material of interest.

Fluorescence measurements have been taken. The wavelengths used wereblue-430±30 nm and green-525±15 nm. The LEDs were driven directly fromthe signal generator (no other power input required) and oscillated at10 kHz and 200 mV amplitude, peak to peak. Fluorescence signalinterference was detected via custom photodiode shields and photodiodesthat have peak sensitivity at the expected emission range(s).Differentiation of fluorescence measurements versus background versusenvironmental illumination are dealt with by a combination of the customcoloured shields and the fact that the LEDs and photodiodes are housedon the inner face of the integrating sphere. In this example, the shieldused on one photodiode was green (525±15 nm) and on the other photodiodethe shield used was red (630±18 nm). These shields were selected toallow detection of the fluorescence output of the stain nile red when itis exposed to a lipid rich environment.

Whilst the integrating spheres of FIGS. 10 and 11 are shown with onlyinternal light sources, they could be combined with the arrangement ofFIG. 1, so that internal and external sources could be used. Preferably,the internal sources are used for fluorescence measurements, asdescribed above. Preferably, the external source is used for otheroptical measurements, as described above.

The present invention has numerous applications. For example, theinvention can be used to establish early growth of pathogens inhuman/animal/food samples or on medical devices such as drips. It canalso be used to detect minute changes in cellular concentrations inchemotherapy studies for microbiology/oncology/mycology or to detectimpurities in water or other fluids.

As another example, the invention can be used for simple cell counting.Enumerating the number of cells in a sample is a common microbiologicaltask and the invention makes it simple, rapid and easy and with thepossibility of an operator being able to build a database of their owncells in a particular medium to allow for the rapid detection of smallchanges in a sample, such as a burgeoning contamination or a smallcolour change in the medium. Using the invention the number of bacteriain a sample can be determined with precision down to a lower limit of˜10 microbes per mL.

The invention is sensitive enough to be able to differentiate betweencultures with very similar cell numbers. In particular, the inventionallows for rapid drug susceptibility testing of species or strains ofbacteria/fungi to establish the level of drug which will kill or inhibitgrowth of a given organism. For example, small shifts in cell numberearly in drug susceptibility studies can be detected where one culturehas been dosed with a bacteriostatic concentration of an antibiotic andanother is allowed to replicate naturally (as demonstrated above withreference to FIGS. 7 to 9).

The invention could also be useful in determining the cell state of abacterial culture. This is because some microbes change theirmorphologies under different circumstances, and different sizes andshapes of bacteria will scatter light differently. Equally, MIC (minimuminhibitory concentration—the smallest amount of a given drug that willinhibit the growth of a given bacterial species of strain)/MBC (minimumbactericidal concentration—the smallest amount of a given drug that willkill all present cells of a given bacterial species of strain in asample) breakpoint analysis can be done to establish the point at whicha microbial strain is or is not responding to a particular antibiotic orcombination of antibiotics.

In yet another application, the growth of microbes in a non-opaque mediacan be tracked. This can be done in a range from the lower detectionlimit (<10 microbes per mL) to ˜10⁹ microbes per mL as a function oftime. This can be automatic at distinct time intervals or manual at theoperator's discretion, or a combination of the two.

Using post-acquisition data analysis the growth rate of microbialsamples can be determined, i.e. the time it takes for bacteria todivide. Also in certain assays (e.g. drug susceptibility) the number ofbacteria can be estimated. This is done using an automated analysis andso can provide systematic computation without user input into theanalysis.

As an example, FIG. 12 shows growth curves from M. smegmatis. In FIG.12, the different curves represent different dilutions. The circlesrepresent the raw data points. The solid line represents the fittedgompertz function. The Y axis is the log base 2 of the value of thescattering intensity. FIG. 13 is an example of a fitted gompertzfunction. Mu is the gradient at the steepest part of the curve. Time topositivity (TTP) is where this line intercepts the x-axis. Mu is used tocalculate the growth rate of the bacterium. TTP is used to calculate thenumber of bacteria.

FIG. 14 shows the relationship between 1 over Mu (the steepest part ofthe gompertz function) and the time it takes for the bacteria to divide.This allows for conversion between the Mu value from the gompertz curveand an estimate of the time it takes the bacteria to divide. Therelationship between Mu and the Time per generation/division can beexpressed as:estimate of time per division=−1.37578/Mu*1.1912

Hence, by measuring Mu, an estimate of the time per division can beestimated.

Using the above equation, it can be shown that:Number of division till positivity=TTP/Estimate of time per division.

FIG. 15 is a plot of “Number of divisions till positivity” vs number ofcells that were in the sample at the start as derived by CFUs (colonyforming units). FIG. 15 applies to cultures that only have exponentialgrowth, i.e. they have no lag phase. The when the time to positivity isdivided by the estimated time per division (as derived in FIG. 14) theresult is the number of divisions to positivity. This is represented onthe y axis. This graph shows the relationship between the number ofdivisions to positivity and starting bacterial concentration. Thisallows starting bacterial concentration to be estimated using the valuesgenerated from the gompertz function.

Using the invention, any variation from the norm in a fluid can bedetected, including a colour change due to either suspended colloidalparticles or chemical reaction. A change away from transparency towardsthe red end of the spectrum will cause absorption of more red light, sochanging the detection parameters. The same holds true towards the blueend of the spectrum but the detection parameters will be altereddifferently allowing differentiation and detection. Adding differentcoloured lasers boosts this capacity.

A skilled person will appreciate that variations of the disclosedarrangements are possible without departing from the invention. Forexample, although the main area of application described above relatesto medical analytics, other applications are possible. For example,because the device can detect any particle in a non-opaque liquid, itcould be used to find any particle in a liquid medium, such as dust,sand or grit in fluids such as high-quality bottled water. It could alsobe used to test fruit juices being imported, as they need to prove theyare not carrying non-endemic bacteria or fungal spores. To do this, athreshold of scattering intensity can be used as a blank and anyvariation from this can be registered and recorded as a difference whichhas process significance. Accordingly, the above description of aspecific embodiment is made by way of example only and not for thepurposes of limitations. It will be clear to the skilled person thatminor modifications may be made without significant changes to theoperation described.

The invention claimed is:
 1. A system for measuring at least oneproperty of a liquid sample comprising biological material, the systemcomprising: an integrating light collector comprising a cavity in whichthe liquid sample is at least partially contained, an entry port forallowing light to enter the cavity, an exit port to allow un-scatteredlight to pass to a beam dump, a first part comprising a sample port, anda second part separable from the first part and comprising a reflectivesurface that is configured to reflect light and thereby enable theintegrating light collector to collect light scattered by interactionwith the liquid sample; a sample holder configured for containing theliquid sample comprising biological material within its internal volumeand for at least partially containing the liquid sample in theintegrating light collector, wherein the sample holder is inserted intothe sample port such that the first part, the second part, and a portionof the sample holder form the cavity in which the liquid sample is atleast partially contained; a light source configured to introduce lightin the integrating light collector such that the light is incident onthe liquid sample at least partially contained in the cavity; a signalgenerator or modulator configured to apply a known modulation of thelight output by the light source; a phase-sensitive detector comprisinga lock-in amplifier, wherein the phase-sensitive detector is configuredto detect scattered modulated light that is collected in the integratinglight collector; and a processor configured to analyse the scatteredmodulated light detected by the phase-sensitive detector at a first timeand the scattered modulated light detected by the phase-sensitivedetector at a second time and determine a change in the detectedmodulated light as a function of time thereby to determine at least oneproperty of the liquid sample selected from: drug susceptibility of thebiological material; a change in a number of cells in the liquid sample;a change in cell state; and a change in the biological material.
 2. Asystem as claimed in claim 1, wherein the light source comprises a laseror LED.
 3. A system as claimed in claim 1, wherein the light source hasa wavelength in the range of 590 nm to 650 nm.
 4. A system as claimed inclaim 1, wherein the light source has a wavelength in the range of 620nm to 750 nm.
 5. A system as claimed in claim 1, wherein the known lightmodulation is at least one of a phase modulation, a frequencymodulation.
 6. A system as claimed in claim 1, wherein the sample holderis configured to hold a sample container containing at least part of theliquid sample substantially within the integrating light collector.
 7. Asystem as claimed in claim 6, wherein the sample container comprises asample cuvette.
 8. A system as claimed in claim 1, wherein thephase-sensitive detector is provided on an internal surface of theintegrating light collector and the system further comprises means forpreventing un-scattered light from being detected by the phase-sensitivedetector.
 9. A system according to claim 1, wherein the processor isconfigured to analyze to determine at least the change in the biologicalmaterial, wherein the change in the biological material is a change in alevel or concentration of a pathogen in the biological material.
 10. Asystem according to claim 1, wherein the biological material comprisesat least one of: a species of strain of bacteria or fungi; a microbe.11. A method for monitoring a biological material in a liquid sample,the method comprising: introducing the liquid sample into an integratinglight collector thereby to at least partially contain the liquid sample;illuminating the liquid sample in the integrating light collector withlight having a known modulation, so that the light passes through and isscattered by the liquid sample; detecting scattered modulated lightcollected in the integrating light collector, wherein the integratinglight collector comprises a cavity in which the liquid sample is atleast partially contained, an entry port for allowing light to enter thecavity, an exit port to allow un-scattered light to pass to a beam dump,a first part comprising a sample port, and a second part separable fromthe first part and comprising a reflective surface that is configured toreflect light and thereby enable the integrating light collector tocollect light scattered by interaction with the liquid sample, andwherein the introducing the liquid sample into the integrating lightcollector includes inserting the sample holder into the sample port suchthat the first part, the second part, and a portion of the sample holderform the cavity in which the liquid sample is at least partiallycontained, wherein detecting scattered modulated light collected in theintegrating light collector comprises detecting first scatteredmodulated light that is scattered in the integrating light collector ata first time and detecting second scattered modulated light that isscattered in the integrating light collector at a second time, andwherein the method further comprises: analysing the detected firstscattered modulated light and the detected second scattered modulatedlight to determine a change in the detected modulated light as afunction of time thereby to determine at least one of: drugsusceptibility of the biological material; a change in a number of cellsin the liquid sample a change in cell state; and a change in thebiological material.
 12. A method as claimed in claim 11, wherein themethod comprises analyzing to determine at least the change in thebiological material, wherein the change in the biological material is achange in a level or concentration of a pathogen in the biologicalmaterial.
 13. A method as claimed in claim 11, further comprisingmonitoring drug susceptibility by introducing a drug into the liquidsample and analysing to determine drug susceptibility.
 14. A method asclaimed in claim 11, wherein the liquid sample comprises at least one ofa species or strain of bacteria/fungi; a microbe.
 15. A device formeasuring at least one property of a liquid sample comprising biologicalmaterial, the system comprising: an integrating light collectorcomprising a cavity in which the liquid sample is at least partiallycontained, an entry port for allowing light to enter the cavity, an exitport to allow un-scattered light to pass to a beam dump, a first partcomprising a sample port, and a second part separable from the firstpart and comprising a reflective surface that is configured to reflectlight and thereby enable the integrating light collector to collectlight scattered by interaction with the liquid sample; a sample holderconfigured for containing the liquid sample comprising biologicalmaterial within its internal volume and for at least partiallycontaining the liquid sample in the integrating light collector, whereinthe sample holder is inserted into the sample port such that the firstpart, the second part, and a portion of the sample holder form thecavity in which the liquid sample is at least partially contained; alight source configured to introduce light in the integrating lightcollector such that the light is incident on the liquid sample at leastpartially contained in the cavity; a signal generator or modulatorconfigured to apply a known modulation of the light output by the lightsource; and a phase-sensitive detector comprising a lock-in amplifier,wherein the phase-sensitive detector is configured to detect scatteredmodulated light that is collected in the integrating light collector,wherein detecting scattered modulated light collected in the integratinglight collector comprises detecting first scattered modulated light thatis scattered in the integrating light collector at a first time anddetecting second scattered modulated light that is scattered in theintegrating light collector at a second time.
 16. A system as claimed inclaim 1, wherein at least one of the first part and the second partincludes a port for the light source and a port for the phase-sensitivedetector.